N-Iinked glycoproteins and the pathways that produce them are integral in human health and disease since these glycoconjugates play fundamental roles in virtually all aspects of cellular function. Therefore, a detailed, molecular understanding of the processes and factors that regulate glycoconjugates in humans and the pathogenic microorganisms that threaten human health is of utmost importance. The focus of this proposal is on N-linked glycosylation in the pathogenic prokaryote Campylobacter jejuni. This is a highly relevant target for detailed investigation since studies will provide tools and approaches for understanding the role of Nlinked glycosylation in microbial pathogens. In addition, this pathway represents an experimentally tractable analog that would offer insights and new methodological approaches for deriving information on the more complex eukaryotic systems. In C. jejuni, N-Iinked glycosylation comprises three sequential phases. In the first phase, a di- N-acetylbacillosamine donor (UDPdiNAcBac) is biosynthesized from a common carbohydrate precursor (UDPGlcNAc). This glycosyl donor, together with the more common UDP- Nactylgalactosamine and UDP-glucose, provide the building blocks for the next phase of enzymes. In this phase, a series of membrane-associated transformations, involving the action of a phosphoglycosyl transferase and several glycosyl transferases, afford the obligate membranebound polyprenylpyrophosphate-linked heptasaccharyl donor for protein glycosylation. The pathway culminates with glycan transfer to an asparagine side chain in an intact protein, which is catalyzed by the integral membrane-bound enzyme oligosaccharyl transferase (OTase). In this program we will implement chemical and physical approaches to develop new knowledge about the N-linked glycosylation pathway in C. jejuni. This knowledge will then provide important insight into the role of N-linked glycosylation in bacterial pathogens and the function of the more complex eukaryotic pathway that is central to a broad range of physiological functions ranging from development to the immune response.